Aberrant glycosylation is a typical hallmark of cancer cells. Carbohydrate tumor antigens on glycoproteins and glycolipids are therefore targets for active and passive immunotherapy. These highly abundant antigens are de novo expressed or upregulated due to changes in the complex glycosylation apparatus of tumor cells. Various lipid or protein bound carbohydrate tumor antigens are described, e.g. GM2, GD2, GD3, fucosylated GM1, Globo H, Ley and the mucin core structures Tn, Sialyl-Tn and the Thomson Friedenreich antigen.
Thomsen-Friedenreich antigen (TF) is a known carbohydrate structure described as a tumor antigen in a series of reports. TF exists in two forms, TF alpha and TF beta, which can be linked to proteins or glycolipids.
Core-1 is the disaccharide Gal-β1,3-GalNAc which is in particular O-glycosidically linked in an alpha-anomeric configuration to the hydroxy amino acids serine or threonine of proteins in carcinoma cells. Core-1 corresponds to the TF-alpha structure of Thomsen-Friedenreich and is linked only to proteins on tumors. Hence, the terms core-1 and Thomsen-Friedenreich do not necessarily refer to identical structures.
Core-1 is masked by other carbohydrate components in healthy and benign-diseased tissue but is uncovered in a majority of carcinomas and in some non-epithelial malignancies. Therefore, core-1 is a specific pan-carcinoma antigen.
Core-1 is an important tumor antigen. Core-1 is expressed on over 60% of primary colon carcinomas and over 90% of liver metastases from colon cancer as well as on the majority of the carcinomas of other major indications including breast, lung, ovarian, prostate, and other gastrointestinal cancers such as gastric, and pancreatic carcinomas. Core-1 is an independent prognostic marker for patients with colon carcinomas, the mortality rate increases and the medium survival decreases in accordance with the increasing intensity of core-1 expression. The development of liver metastases correlates with the expression of core-1. Patients with core-1 positive primary carcinomas develop liver metastases in nearly 60% of the cases, while the risk for liver metastasis with core-1-negative tumors is significantly lower (less than 20%). Besides mediating metastasis into the liver core-1 may also play a role in the metastasis via the endothelium.
The exceptionally high pan-carcinomic specificity, prognostic relevance and direct involvement in liver metastasis render Thomsen-Friedenreich and particularly core-1 a prime target for cancer immunotherapy.
As a result of the wide distribution of the core-1, it is advantageous to provide a core-1 positive microorganism having a cell surface structure corresponding to core-1 which can be used as pharmaceutical, for example, in the prevention and treatment of cancer, in particular for vaccination.
There were attempts to provide a therapy approach based on Thomsen-Friedenreich. E.g. Shigoeka et al. (1999) describe the inhibition of liver metastasis from neuramidase treated Colon 26 cells by an anti-Thomsen-Friedenreich specific monoclonal antibody in a mouse model. However, due to the difficulties in generating highly specific anti-TF antibodies and because of their nature as IgM isotypes with comparably lower intrinsic affinities of single binding domains, TF-specific antibodies were not further developed so far. Further, some anti-TF-antigen antibodies are not clinically useful because they cause undesirable proliferation of tumor cells. Also WO 2006/012626 describes the therapeutic use of anti-TF antigen antibodies. Binding of TF-specific antibodies has been shown to inhibit the proliferation of tumor cells (Jeschke et. al. (2006)).
Furthermore, there were also attempts to develop vaccines based on Thomsen-Friedenreich. Most of them focused on the induction of antibody responses. E.g. Livingston and Lloyd (2000) used non-natural TF-conjugates, wherein synthetic TF was randomly coupled to KLH. This conjugates raised a humoral immune response against synthetic TF but not against TF on natural ligands (Adluri et al. (1995)). They were thus not TF specific as they would not recognize TF on a tumor structure.
Springer and Desai used vaccination with a T/Tn vaccine composed of types 0 and MN red blood cell derived glycoproteins which resulted in improved breast cancer patient survival, although only small amounts of IgM were made. However, IgM represents a less mature immune response and many previous studies relating to antibodies to TF-Ag involve IgM antibodies, therefore more pronounced highly TF specific immune responses would be needed and preferably an IgG response.
Few reports are known which describe microorganisms supposedly positive for TF. E.g. Springer et al. (Brit J Haematol 47 (1981), 453-460; Transfusion 19 (1979), 233-249) report on an aerobic microorganism (E. coli 086) which can generate a polyclonal antibody response in chickens and humans which might also recognize TF on human erythrocytes. Springer used adsorption of anti-T and hemagglutination assays with sialidase-treated T erythrocytes in order to determine roughly the specificity of the immune response. However, sialidase-treatment of human erythrocytes results in demasking of several carbohydrate epitopes, among them but not exclusively TF and in particular core-1. Therefore, the reaction tested by Springer does not show a specificity for TF and in particular core-1 due to cross-reactivities. A respective non-specific microorganism has only a limited suitability as a vaccine due to its unspecificity as it would not raise a strong immune response which is specifically directed against TF but against similar TF-like structures and hence potentially also increasingly against non-tumor tissues or cells of the body. Furthermore, tests have shown that E. coli 086 is not positive for core-1 expression because it is not bound by core-1 specific antibodies.
WO 2008/055703 discloses core-1 positive microorganisms, in particular, microorganisms of the species Bacteroides ovatus expressing a core-1 antigen on their cell surface such as AG6 and MU1 and their use as pharmaceuticals. Expression of a core-1 antigen was confirmed by binding of core-1 specific antibodies to said microorganisms. Furthermore, WO 2008/055703 discloses the use of respective microorganisms in therapy.
When using core-1 positive microorganisms as pharmaceuticals it is desirous that the core-1 positive microorganism not only expresses a core-1 antigen, but that the microorganism shows a high core-1 antigen expression in order to effectively stimulate a core-1 specific immune response. Furthermore, it is desirous that the core-1 positive microorganism that is chosen for use as pharmaceutical has a stable, and preferably homogeneous high core-1 antigen expression. A stable, high core-1 antigen expression ensures that the product containing the core-1 positive microorganism exhibits less variability what is important for the product quality, in particular in the pharmaceutical field. A stable, high core-1 antigen expression is also important in order to fulfill the regulatory requirements.
Therefore, it is the object of the present invention to provide alternative core-1 positive microorganisms, in particular microorganisms having a stable, high core-1 antigen expression on their cell surface.